Utilization of the recombinant DNA technology in an attempt to produce heterologous peptides or proteins fails in large-scale production in many instances even in the case of direct expression of the desired gene products. As for the reasons therefor, it has been pointed out, for instance, that the translation initiation reaction on the genes to be expressed is inhibited by the influence of the higher-order structure of mRNA in the vicinity of the initiation codon [D. Iserentant, E. Fiers, Gene, 9, 1 (1980)] and that when the desired gene products have a low molecular weight, they are readily degraded particularly by host-derived proteases.
The fusion protein method has now attracted attention as a method capable of solving these problems. In this fusion protein method, desired peptides or proteins are produced in a form fused with a carrier, for example a peptide or protein, which is stable in the host and is produced in large quantities. This fusion protein method includes those cases as well in which carriers don't come under the category of proteins in general, namely carriers composed of less than 50 amino acid residues. Even in the case of direct expression as fusion proteins, the processing by methionine amino-peptidase in the host is incomplete, so that the methionine residue derived from the initiation codon is not fully eliminated but partly remains, giving, in many instances, the desired gene products with methionine added to the N terminus thereof. A possibility has been pointed out that such gene products with said methionine added may have an antigenicity problem (Japanese Patent Laid-open No. 171699/1987). Therefore, for obtaining desired peptides or proteins from such fusion proteins or methionine-retaining gene products, it is necessary to excise the desired portions from the fusion proteins or such gene products. Chemical and enzymatic methods are known for this excision.
Chemical methods that are known include, for example, the cleavage of the peptide bonds on the C-terminal side of Met by cyanogen bromide [D. V. Goeddel et al., Proc. Natl. Acad. Sci. USA, 76, 106-110 (1979)], the cleavage of the peptide bonds on the C-terminal side of Trp by BNPS-skatole or N-chlorosuccinimide (NCS) [Y. Saito et al., J. Biochem., 101, 123-134 (1987)], the cleavage of the peptide bond between Asp-Pro by an acid, for example, 70% formic acid or the like [Biochem. Biophys. Res. Commun., 40, 1173 (1970)], and the cleavage of the peptide bond between Asn-Gly by hydroxylamine.
However, because of strong dependency on the structure of each substrate fusion protein, the cleavage reactions, except for the cleavage by cyanogen bromide, give low yields and readily lead to side reactions. The cleavage with cyanogen bromide is widely used but cannot be applied to the cases where the desired gene products contain Met. When the amino acid next to Met is Ser or Thr, the cleavage reaction with cyanogen bromide scarcely proceeds in some instances. When the cleavage is effected between Asp-Pro or Asn-Gly bond, both the N-terminal and C-terminal sides of the peptides or proteins resulting from the cleavage have one or more remaining amino acids, so that it is difficult to obtain gene products having desired amino acid sequences at the N and C termini by cleavage.
Enzymatic methods that are known include, among others, the cleavage using a protease showing strict primary specificity, namely specificity to an amino acid just before (P1 position) or behind (P1' position) the peptide bond to be cleaved, for example the cleavage of the peptide bond on the C-terminal side of Arg or Lys by trypsin or a trypsin-like enzyme such as endoproteinase Arg-C [J. Shine et al, Nature, 285, 456-461 (1980)], the cleavage of the peptide bond on the C-terminal side of Lys by lysyl endopeptidase or endoproteinase Lys-C (Japanese Patent Laid-open No. 275222/1986), and the cleavage of the peptide bond on the C-terminal side of Glu or Asp by an enzyme specific to acidic amino acids, for example V8 protease (Japanese Patent Laid-open No. 501391/1985).
Generally, however, these Proteases are effective only when any amino acid recognized by them is not contained in the desired gene products. Therefore they are applicable only to a very limited range of gene products.
Not only enzymes showing primary specificity but also enzymes showing strict secondary specificity, namely specificity to sequence around the peptide bond to be cleaved, are in use. For example, reports are available on the cleavage of the peptide bond between X-Gly of chicken pro .alpha.-2 Collagen or Pro-X-Gly-Pro (SEQ ID NO: 17) by a collagenase [Japanese Patent Publication No. 44920/1987; J. Germino et al., Proc. Natl. Acad. Sci. USA, 81, 4692-4696 (1984)], the cleavage of the peptide bond on the C-terminal side of Ile-Glu-Gly-Arg (SEQ ID NO: 20) by blood coagulation factor Xa (Japanese Patent Laid-open No. 135591/1986), the cleavage of the peptide bond on the C-terminal side of Gly-Pro-Arg etc. by thrombin (Japanese Patent Laid-open No. 135500/1987), the cleavage of the peptide bond on the C-terminal side of Phe-Arg with kallikrein (Japanese Patent Laid-open No. 248489/1987), the cleavage of the peptide bond on the C-terminal side of Val-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 21) by entero-peptidase [T. P. Hopp et al., Biotechnology, 6, 1204-1210 (1988); Japanese Patent Laid-open No. 166200/1981], the cleavage of the peptide bond between Leu-Leu of Pro-Phe-His-Leu-Leu-Val-Tyr (SEQ ID NO: 22) by renin (Japanese Patent Laid-open No. 262595/1985), the cleavage of the peptide bond on the C-terminal side of poly-Gly by lysostaphin (Japanese Patent Laid-open No. 160496/1989), the cleavage of the peptide bond on the C-terminal side of ubiquitin by ubiquitin-N.sup..alpha. -protein hydrolase [T. R. Butt et al., Proc. Natl. Acad. Sci. USA, 86, 2540-2544 (1989)], and the cleavage of the peptide bond on the C-terminal side of Glu-Gly-Arg by urokinase (Japanese Patent Laid-open No. 100685/1990).
However, the digestion with collagenase or renin allows peptides to remain on both the N-terminal and C-terminal sides of the cleavage site and therefore makes it difficult to obtain gene products having the desired amino acid sequences. It is reported that many enzymes, in particular thrombin, cause cleavage also at other sites than recognition sequences and are lacking in general-purpose property because of their low specificity [e.g. J. Y. Chang, Eur. J. Biochem., 151, 217-224 (1985)]. Blood coagulation factor Xa is currently in widest use for excising desired gene products from fusion proteins. Cases are, however, reported in which it cleaves other sites than specific recognition sequences as well, so that desired gene products cannot be obtained [e.g. S. Nishikawa et al., Protein Engineering, 1, 487-492 (1987)]; hence factor Xa is lacking in general-purpose property. For instance, when blood coagulation factor Xa or kallikrein is used for the excision of vasoactive intestinal polypeptide (hereinafter abbreviated as VIP) derivative as the desired gene products from fusion proteins, cleavage occurs within VIP, for example between Arg.sup.14 -Lys, so that no VIP can be obtained. Furthermore, these proteases are required in considerable quantities, for example 0.1 to 0.001 mole, generally about 0.01 mole, per mole of substrate fusion protein.
Another method of producing desired gene products is also known which comprises joining a carrier sequence to a DNA sequence coding for a desired gene via a sequence coding for a processing signal which is a dipeptide, thus producing a fusion protein, and enzymatically cleaving the dipeptide. Thus, reports are available on the production of desired gene products utilizing such dipeptide, in particular a pair of basic amino acid residues, for example the secretory production of desired gene products using the preproleader sequence (containing Lys-Arg) of .alpha. mating factor produced by Saccharomyces cerevisiae (Japanese Patent Laid-open No. 132892/1984) and the secretory production of desired gene products utilizing the preproleader sequence of .alpha. mating factor of Kluyveromyces lactis (Japanese Patent Laid-open No. 124390/1989). In these methods, fusion proteins undergo limited proteolysis in vivo and desired gene products are secreted extracellularly.
However, these methods are only applicable to those cases in which specific hosts, for example Saccharomyces cerevisiae, Kluyveromyces lactis and the animal cell line AtT-20, which possess processing systems for basic amino acid pair cleavage, secretion, glycosylation and so forth; hence, they are not generally applicable methods. The use of these systems re sults in various problems, for example formation of incompletely processed gene products, namely gene products having a preproleader-derived Glu-Ala sequence added (e.g. Japanese Patent Laid-open No. 132892/1984; Japanese Patent Laid-open No. 502661/1987), and generally low production efficiency and low productivity because of various processing steps, such as proteolysis and glycosylation steps, being rate-limiting steps.